Evaluation of O₃ Effects on Cumulative Photosynthetic CO₂ Uptake in Seedlings of Four Japanese Deciduous Broad-Leaved Forest Tree Species Based on Stomatal O₃ Uptake

Abstract

The current level of tropospheric ozone (O₃) is expected to reduce the net primary production of forest trees. Here, we evaluated the negative effects of O₃ on the photosynthetic CO₂ uptake of Japanese forest trees species based on their cumulative stomatal O₃ uptake, defined as the phytotoxic O₃ dose (POD). Seedlings of four representative Japanese deciduous broad-leaved forest tree species (Fagus crenata, Quercus serrata, Quercus mongolica var. crispula and Betula platyphylla var. japonica) were exposed to different O₃ concentrations in open-top chambers for two growing seasons. The photosynthesis–light response curves (A-light curves) and stomatal conductance were measured to estimate the leaf-level cumulative photosynthetic CO₂ uptake (ΣP_{n_est}) and POD, respectively. The whole-plant-level ΣP_{n_est} were highly correlated with the whole-plant dry mass increments over the two growing seasons. Because whole-plant growth is largely determined by the amount of leaf area per plant and net photosynthetic rate per leaf area, this result suggests that leaf-level ΣP_{n_est}, which was estimated from the monthly A-light curves and hourly PPFD, could reflect the cumulative photosynthetic CO₂ uptake of the seedlings per unit leaf area. Although the O₃-induced reductions in the leaf-level ΣP_{n_est} were well explained by POD in all four tree species, species-specific responses of leaf-level ΣP_{n_est} to POD were observed. In addition, the flux threshold appropriate for the linear regression of the responses of relative leaf-level ΣP_{n_est} to POD was also species-specific. Therefore, species-specific responses of cumulative photosynthetic CO₂ uptake to POD could be used to accurately evaluate O₃ impact on the net primary production of deciduous broad-leaved trees.

1. Introduction

Tropospheric ozone (O₃) has detrimental effects on vegetation [1,2,3]. Current O₃ levels adversely affect growth and physiological functions, such as the photosynthesis of forest tree species [4,5]. Because of this phytotoxicity, net primary production (NPP) and biomass accumulation by temperate forests were estimated to be reduced by 1–16% [6]. Therefore, O₃ indirectly affects radiative forcing through its adverse effects on photosynthetic CO₂ uptake [7]. Despite robust evidence for the negative impact of O₃ on plant productivity, the indirect effect of O₃ on radiative forcing was not mentioned in the Intergovernmental Panel on Climate Change Fifth Assessment report because of a lack of corroborating studies [8]. In Asia, an increase in the surface O₃ concentration is expected, although the changes in its concentration depend on the emission scenario [9,10,11]. In Japan, despite a decrease in the concentrations of O₃ precursors, the surface O₃ concentration has been increasing because of a reduction in NO titration and an increase in transboundary air pollution [12]. Therefore, it is necessary to quantify the negative impact of O₃ on the photosynthetic CO₂ uptake of Japanese forest tree species to account for the indirect effect of O₃ on radiative forcing.

Ozone enters leaves through their stomata and damages their cellular components [13,14,15]. Because the real impacts of O₃ on plants mainly depend on the amounts of O₃ reaching the sites of damage within the leaves, these impacts can be quantified by measuring the cumulative stomatal O₃ uptake, defined as phytotoxic O₃ dose (POD) and associated response functions [16,17,18]. Because the degree of stomatal opening adjusts continuously to environmental changes [19], this O₃ flux-based approach requires the development of mathematical models to estimate the POD in leaves, using stomatal responses to environmental factors [20,21,22]. In Japan, several researchers have established multiplicative models to estimate the POD in the leaves of forest tree species [23,24,25,26,27]. In addition to modeling stomatal O₃ uptake, the degree of the O₃-induced reduction in photosynthetic CO₂ uptake should be evaluated, based on the POD, to quantify the negative impact of O₃ on the NPP.

According to a meta-analysis by Wittig et al. (2007) [28], the light-saturated net photosynthetic rate (A_sat) decreases at a rate of 0.22% per mmol O₃ m⁻² leaf area (LA) as POD increases. Because the relationship between POD and the percent change in A_sat varies with species and genotypes within species, as well as with locations and experimental conditions, little of the variability in the degree of percent change in A_sat is accounted for by the POD [28]. In addition to this variability, seasonal variation in the degree of O₃-induced reduction in A_sat could explain why POD is responsible for only a small percentage of the changes in A_sat measured at a certain time. For example, the degree of O₃-induced reduction in A_sat of Populus nigra is lower in autumn than in summer [29], whereas POD is greater in autumn than in summer owing to the longer accumulation period. Because POD could be related to cumulative O₃ damage, a cumulative O₃ effects on net photosynthesis is an appropriate dependent variable in the response function of photosynthetic CO₂ uptake to POD. Therefore, it is necessary to evaluate the degree of O₃ damage on the cumulative photosynthetic CO₂ uptake at leaf-level, which could be easily incorporated into models of the carbon cycling, throughout an entire growing season, taking into consideration of seasonal and diurnal changes in the O₃ damage on A_sat.

In Japan, temperate deciduous forests have been classified into three forest types: cool temperate deciduous forests, warm temperate deciduous forests and cool temperate mixed deciduous broadleaf/conifer forests [30]. The dominant tree species are Fagus crenata, Quercus serrata and Quercus mongolica var. crispula, respectively [30] and Betula platyphylla var. japonica is a typical early successional species in these forest types [31]. There are considerable differences in the sensitivities of these tree species to O₃ concentration, as evaluated by whole-plant growth [32,33]. Although a stomatal conductance model to estimate the PODs in the leaves of these tree species has been established [27], the degree of O₃ damage on cumulative photosynthetic CO₂ uptake has not been evaluated based on the POD. Therefore, in the present study, we conducted an experimental study on the effects of O₃ on cumulative photosynthetic CO₂ uptake in the leaves of four representative Japanese deciduous broad-leaved tree species with different O₃ sensitivities. To account for seasonal and diurnal changes in the degree of the O₃-induced reduction in net photosynthetic rate when estimating the cumulative O₃ effects on photosynthetic CO₂ uptake, we measured photosynthesis–light response curves (A-light curves) monthly throughout an entire growing season.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

In December 2011, 3-year-old seedlings of F. crenata and 2-year-old seedlings of Q. serrata, Q. mongolica var. crispula and B. platyphylla var. japonica were individually planted in 1/2000 Wagner’s pots (volume: 12 L, diameter: 240 mm, depth: 258.5 mm) filled with commercial horticultural soil (Takii & Co., Ltd., Kyoto, Japan). The N, P and K contents in the potted soil were 320, 210 and 300 mg L⁻¹, respectively. On 25 April 2012, the seedlings were transferred into 12 rectangular open-top chambers (OTCs, 13.0 m² of growth space and 2.4 m in height) located at the Akagi Testing Center of the Central Research Institute of Electric Power Industry (Maebashi, Gunma, Japan) and eight seedlings per species were grown in each chamber. To measure the initial whole-plant dry mass (WDM), including belowground parts, the 10 seedlings of each species, which were not assigned to the chamber and whose mean height and stem base diameter were similar with those of the seedlings assigned to the chambers, were harvested, dried at 80°C in an oven for more than 5 days and weighed. The initial WDMs of F. crenata, Q. serrata, Q. mongolica var. crispula and B. platyphylla var. japonica seedlings were 11.1 ± 5.2 g, 36.9 ± 8.1 g, 23.7 ± 5.51 g and 11.1 ± 1.8 g, respectively (±SD, n = 10). The latitude, longitude and elevation above sea level at the experimental site are 36°28′ N, 139°11′ E and 540 m, respectively. In late August 2012 and in late April and late July 2013, the seedlings were rotated among and inside the OTCs to minimize variation among the OTCs for each treatment and among the seedlings within the OTC owing to chamber and positional effects, respectively. All the seedlings were regularly irrigated to keep the potted soil moist. On 12 April and 5 July 2013, all the seedlings were fertilized with 8 g m⁻² of a slow-release solid fertilizer (N:P:K = 8:8:8, Ohmiya Green Service Co., Ltd., Saitama, Japan) to avoid the nutrient deficiency, which could limit physiological processes. The air temperature, relative air humidity and photosynthetic photon flux density (PPFD) inside the OTCs were monitored during the experimental period from 26 April 2012 to 14 November 2013 (Table S1). The vapor pressure deficit (VPD) was calculated from the saturated vapor pressure, which was calculated from air temperature and the relative air humidity.

2.2. Gas Treatment

From 26 April to 22 November 2012 and from 29 March to 14 November 2013, the seedlings were exposed to charcoal-filtered (CF) air or O₃ at 1.0, 1.5 or 2.0 times the ambient concentration in the OTCs. For each treatment, three chamber replications, for a total of 12 chambers, were used. The ambient O₃ concentration was independently monitored by a UV absorption O₃ analyzer (ML9810, Teledyne Monitor Labs, Englewood, CO, USA) at the experimental site. Ozone was generated from oxygen-enriched dry air by an electrical discharge O₃ generator (Oz-24-UA, Ebara Corporation, Japan) and injected into the OTCs through a water trap that removed the nitrogen byproducts produced by the O₃ generator [34]. The O₃ concentrations at 90 cm above the OTC floors were monitored using the UV absorption O₃ analyzer. The O₃ concentration accumulated over a threshold of 40 nmol mol⁻¹ (ppb) (AOT40) is used to investigate the concentration-based critical levels for O₃ impact on vegetation. Thus, whether the O₃ concentration in the present study was above the level having negative impact on vegetation, could be investigated, even though the AOT40 is biologically less relevant than POD in O₃ impact assessments on vegetation [18]. Consequently, we calculated AOT40 as the sum of the differences between the hourly mean O₃ concentrations and 40 ppb when the former exceeded 40 ppb during the daylight hours and the solar irradiation was greater than 50 W m⁻².

2.3. Measurement of Stomatal Diffusive Conductance

The stomatal diffusive conductance to water vapor (g_sw, mmol H₂O m⁻² LA s⁻¹) in the leaves of the seedlings was measured using an LI-1600 steady-state porometer (LI-COR, Lincoln, NE, USA). Throughout the O₃-exposure period, the measurements were conducted on the abaxial sides of the 1st flush leaves of F. crenata, the 1st and 2nd flush leaves of Q. serrata and Q. mongolica var. crispula and the early and late leaves of B. platyphylla var. japonica seedlings. Corresponding PPFD, air temperature, relative air humidity, soil volumetric water content (SWC, %) at a 10-cm depth from the surface of the potted soil and O₃ concentration were also recorded. The measurements were conducted throughout the daytime on the representative leaves at a rate of about two days per month during the growing seasons.

2.4. Calculation of POD

The POD with a flux threshold of Y nmol O₃ m⁻² LA s⁻¹ (POD_Y, mmol O₃ m⁻² LA), during one growing season, was calculated according to the method described by CLRTAP (2017) and Kinose et al. (2014) [18,27], with modifications of the parameters in the stomatal conductance model based on the g_sw obtained in the present study. The period used to calculate the POD_Y was from the day of leaf emergence of each leaf type to the day on which the net photosynthetic rate was measured for each growing season. The POD_Y during the daylight hours was calculated by accumulating stomatal O₃ flux (F_st, nmol O₃ m⁻² LA s⁻¹) with or without a flux threshold of Y nmol O₃ m⁻² LA s⁻¹ (POD_Y or POD₀, respectively). The F_st was calculated using the following equation:

$$F_{\mathrm{st}}=\frac{{\left[{\mathrm{O}}_3\right]}_{\mathrm{air}}-{\left[{\mathrm{O}}_3\right]}_{\mathrm{leaf}}}{r_{\mathrm{b}\_\mathrm{ozone}}+r_{\mathrm{s}\_\mathrm{ozone}}}$$

(1)

where [O₃]_air and [O₃]_leaf represent O₃ concentrations in the air and the intercellular space (nmol O₃ mol⁻¹), respectively, and r_{b_ozone} and r_{s_ozone} represent boundary layer resistance and stomatal resistance for O₃ diffusion, respectively. For each gas treatment, the average hourly mean O₃ concentration of three chambers was used for [O₃]_air. The [O₃]_leaf was assumed to be zero [35]. The r_{b_ozone} was calculated from the crosswind leaf dimension (L_d, m) and wind speed (u, m s⁻¹) as follows:

$$r_{\mathrm{b}\_\mathrm{ozone}}=1.3\times 150\times \sqrt{\frac{L_d}{u}}$$

(2)

where the factor 1.3 accounts for the difference in diffusivity between heat and O₃ [18]. Average L_d values for F. crenata, Q. serrata, Q. mongolica var. crispula and the early and late leaves of B. platyphylla var. japonica seedlings were 0.014, 0.015, 0.023, 0.012 and 0.032 m, respectively. The average wind speed 1.5 m above the OTC floors was 0.17 m s⁻¹. The r_{s_ozone}, the reciprocal of the hourly mean stomatal diffusive conductance to O₃ (g_sO3, mmol O₃ m⁻² s⁻¹), was calculated from the hourly mean g_sw and a conversion factor of 0.663 was used to account for the difference in the molecular diffusivity of O₃ and that of water vapor in air [18,36]. The hourly mean g_sw was estimated using the stomatal conductance model reported by Kinose et al. (2014) [27]:

$$g_{\mathrm{sto}}=g_{\max }\times f_{\mathrm{phen}}\times f_{\mathrm{light}}\times \max \left[f_{\min },\left(f_{\mathrm{temp}}\times f_{\mathrm{VPD}}\times f_{\mathrm{SWC}}\times f_{\mathrm{O}3\mathrm{conc}}\right)\right]$$

(3)

where g_sto represents the estimated hourly mean g_sw and g_max represents the species-specific maximum g_sw. The parameters f_phen, f_light, f_min, f_temp, f_VPD, f_SWC and f_O3conc were all expressed in relative terms (i.e., values between 0 and 1) as a proportion of g_max, where f_phen represents the modification of g_max owing to leaf phenological changes; f_light represents the modification of g_max by PPFD (μmol m⁻² s⁻¹); f_min represents the relative value of species-specific minimum g_sw with respect to g_max; f_temp represents the modification of g_max by air temperature (T, °C); f_VPD represents the modification of g_max by VPD (kPa); f_SWC represents the modification of g_max owing to SWC (%); and f_O3conc represents the reduction in g_max resulting from the acute effect of O₃ (ppb). We used the boundary line analysis to define the parameters of each f in the relationships between the ratio of g_sw to g_max and environmental factors [37]. For details of each f and how to analyze the relationships using the boundary line analysis, please refer to Kinose et al. (2014) [27]. Results of the parameterization of stomatal conductance models for the leaves of F. crenata, Q. serrata, Q. mongolica var. crispula and B. platyphylla var. japonica seedlings are shown in Table S3. The POD₀ in the leaves of the tree species during both growing seasons are presented in Tables S2 and S4.

2.5. Measurement of A-Light Curves

From May to November 2012 and from April to October 2013, the A-light curves of the seedlings were measured monthly using an infrared gas analyzer system (LI-6400, LI-COR Inc, Lincoln, Nebraska, US). Two to four seedlings from each chamber were randomly selected to be measured. The seedlings were moved from the OTCs into an experimental room and the measurements were made using three LI-6400s in the room to avoid the influences of weather conditions at the measurement time. The measurements were conducted on one leaf from each leaf type per seedling for the 1st flush leaves of F. crenata, the 1st and 2nd flush leaves of Q. serrata and Q. mongolica var. crispula and the early and late leaves of B. platyphylla var. japonica seedlings. Because the leaf emergence of B. platyphylla var. japonica is heterophyllous [31] and the late leaves emerge successively, the measurements were conducted for the 4th and 5th, 9th and 10th, 15th and 16th (only in 2013) and 18–20th late leaves, as counted from the base of the 1st or 2nd branch from the top of the seedlings. The last measurement day of each growing season was the time when the leaves were almost yellowed at the end of the growing season. When we did not find target leaf for the measurement of A-light curve due to leaf abscission by the exposure to O₃, we did not measure it. The A-light curves were determined at an atmospheric CO₂ concentration of 390 µmol CO₂ mol⁻¹ and a relative air humidity of approximately 60% inside the leaf chamber. As much as possible, the target air temperature inside the leaf chamber was the 10-year average of the monthly mean of the daily maximum air temperature inside the OTCs used in the present study. The actual air temperatures inside the leaf chamber differed among the month and ranged from 17.5 °C to 26.0 °C in 2012 and from 19.0 °C to 28.9 °C in 2013. For detailed information, please refer to the Table S5. To obtain the response of net photosynthetic rate to PPFD, after acclimation of the leaves to light-saturated conditions at a PPFD of 1500 ± 1 µmol m⁻² s⁻¹, PPFD was gradually reduced to zero and the net photosynthetic rates were recorded under PPFDs of 1500, 1,000, 500, 250, 100, 50 and 0 µmol m⁻² s⁻¹. To obtain the A-light curve, a non-rectangular hyperbolic function was fitted to the response of the net photosynthetic rate to PPFD. Although photosynthetic parameters other than the A-light curve could offer a better indication of the overall damage of O₃ on photosynthesis and its mechanisms, we did not measure the parameters, because we focused on the effects of O₃ on the cumulative photosynthetic CO₂ uptake rather than the mechanisms underlying the O₃-induced reduction in net photosynthesis.

2.6. Calculation of Cumulative Photosynthetic CO₂ Uptake

The cumulative photosynthetic CO₂ uptake per unit LA (mol CO₂ m⁻² LA s⁻¹) during each growing season was calculated as the sum of the product of time (1 h) and estimated hourly value of net photosynthetic rate (P_{n_est}, µmol CO₂ m⁻² LA s⁻¹) over one growing season (ΣP_{n_est}, mol CO₂ m⁻² LA). The P_{n_est} was calculated from the hourly mean PPFD monitored inside the OTCs and the A-light curves that were obtained monthly. The PPFD was the mean value of the 12 OTCs. The A-light curves were the means of the three chamber replicates for each treatment. When the hourly mean of PPFD was 0 µmol m⁻² s⁻¹, P_{n_est} was not calculated and was not summed up for the ΣP_{n_est}. The calculation period was from the first to the last measurement days of the A-light curve for each growing season. In each growing season, the A-light curve obtained from the first measurement was used to calculate P_{n_est} during the period from the first measurement day to the middle day of the subsequent measurement day. The A-light curve obtained from the last measurement was used to calculate P_{n_est} during the period from the middle day of the previous measurement day to the last measurement day. The A-light curves, other than those obtained from the first and last measurements in each growing season, were used to calculate P_{n_est} from the middle day of the previous measurement day to the middle day of the subsequent measurement day (approximately 1 month). This calculation assumed the step-changes in the A-light curve between the two measurement days. Because the ΣP_{n_est} is time integral of P_{n_est}, ΣP_{n_est} calculated by assuming linear changes in A-light curve between the two measurement days would result in the same value. When there was no target leaf owing to O₃-induced early leaf abscission (e.g., Tables S6 and S7), we did not accumulate the P_{n_est} for ΣP_{n_est}. The monthly A-light curves of each species are shown in Table S6.

The sums of the product of the number of day and the A_sat at PPFD of 1500 µmol m⁻² s⁻¹ (μmol CO₂ m⁻² LA s⁻¹) over one growing season (ΣA_sat × days, μmol CO₂ m⁻² LA s⁻¹ day) were calculated for each treatment and each growing season as a possible indicator of O₃-induced percent changes in ΣP_{n_est}. The A_sat, which was recorded when the A-light curve was measured and the number of days during the growing season were used to calculate ΣA_sat × days. The periods for the ΣA_sat × days was the same as those for the ΣP_{n_est}. The A_sat measured on the first measurement day was multiplied by the number of days during the period from the first measurement day to the middle of the subsequent measurement day. The A_sat that was measured on the last measurement day was multiplied by the number of days during the period from the last measurement day to the middle of the previous measurement day. The A_sat, other than those measured on the first and last measurement days, were multiplied by the number of days from the middle day of the previous measurement to that of the subsequent measurement (approximately 1 month). The products of the A_sat and the number of days were summed for ΣA_sat × days during each growing season. The monthly A_sat are shown in Table S7. The ΣP_{n_est} and ΣA_sat × days in the leaves of F. crenata, Q. serrata, Q. mongolica var. crispula and B. platyphylla var. japonica seedlings are indicated in Table S4.

2.7. Validation of Cumulative Photosynthetic CO₂ Uptake

To validate the calculation of leaf-level ΣP_{n_est}, leaf-level ΣP_{n_est} was expanded from a LA basis to a whole-plant basis and we analyzed the relationship of the whole-plant-level ΣP_{n_est} with WDM increments over the two growing seasons. The numbers of each leaf type per plant were counted periodically during the growing seasons. Part of each leaf type was sampled to measure the area per leaf using an area meter (LI-3100, Li-Cor Inc, Lincoln, Nebraska, USA). Leaf area per plant of each leaf type was estimated from the product of leaf number per plant and mean area per leaf. For the calculation of the whole-plant-based ΣP_{n_est}, the product of P_{n_est} and estimated leaf area per plant was summed up during each growing season. To measure the WDM at the end of the gas exposure, the seedlings of each tree species were harvested in October and November 2013. The harvested seedlings were dried at 80 °C in an oven for more than 5 days and weighed. The WDM increments over the two growing seasons were calculated using the initial and final WDMs. The effects of O₃ on the WDMs of F. crenata, Q. serrata, Q. mongolica var. crispula and B. platyphylla var. japonica seedlings in October and November 2013 are shown in Table S8.

2.8. Relationships of POD with ΣP_{n_est} and A_sat

For each tree species, the ΣP_{n_est} or A_sat in the CF treatment was used as a reference (100%) to calculate the relative value of ΣP_{n_est} or A_sat (%), respectively, in each gas treatment. It has been suggested that the form of the Weibull function (Y = a × exp [−(X/ω)^λ]) is a biologically realistic response curve through the discussion on the function as a part of the National Crop Loss Assessment Network (NCLAN) in the USA [38]; therefore, this function was fitted to the response of relative ΣP_{n_est} or A_sat to POD without a flux threshold (POD₀). According to the method of CLRTAP (2017) [18], in addition, the response functions of relative ΣP_{n_est} to POD with different flux thresholds (POD_Y) were analyzed using a linear regression analysis to determine any species-specific responses of ΣP_{n_est} to POD. In these analyses, the data obtained from different leaf types (i.e., 1st and 2nd flush leaves or early and late leaves) were pooled for each species, because the responses of the different leaf types were similar. The period for the POD calculation was from the leaf emergence day to each measurement day of A-light curves for A_sat or to the last measurement day of the A-light curves for ΣP_{n_est}.

3. Results

Table 1. Seasonal means of air temperature, vapor pressure deficit (VPD), and photosynthetic photon flux density (PPFD) inside open-top chambers (OTCs) during the O₃-exposure period in 2012 and 2013.

Period	Air Temperature (oC)							VPD (kPa)				PPFD
	Daily Mean		Daily Max.a		Daily Min.b			Daily Mean		Daily Max.a		(mol m−2 day−1)
26 Apr.–22 Nov.,	19.4	(0.1)	25.0	(0.3)	15.2	(0.0)		0.41	(0.03)	1.28	(0.06)	16.9	(0.2)
2012
29 Mar.–14 Nov.,	19.1	(0.1)	25.2	(0.3)	14.6	(0.1)		0.43	(0.04)	1.32	(0.09)	16.8	(0.3)
2013

Each value is the mean value of 12 OTCs; the standard deviation is shown in parentheses. ^a Mean daily 1-h maximum value. ^b Mean daily 1-h minimum value.

shows the air temperature, VPD and PPFD inside the chambers during the O₃-exposure periods from 26 April to 22 November 2012 and from 29 March to 14 November 2013. The climatic conditions inside the OTCs were similar during the exposure periods of 2012 and 2013.

Table 2. O₃ concentrations and AOT40 of O₃ during the O₃-exposure period in 2012 and 2013.

Period	Treatment	Concentration (nl l−1)								Daylight
		Seasonal Mean						Highest		AOT40 b
		24-h Mean		12-h Meana		1-h Max.		1-h Max.		(μl l−1 h)
26 Apr.–22 Nov.,	CF	4.4	(0.4)	4.6	(0.4)	7.4	(0.6)	21.5	(1.5)	0.0	(0.0)
2012 (211 days)	1.0 × O3	25.2	(0.3)	28.1	(0.4)	41.0	(0.7)	111.5	(7.5)	6.0	(0.2)
	1.5 × O3	43.9	(0.7)	46.5	(0.6)	71.2	(0.6)	165.0	(4.0)	33.7	(0.1)
	2.0 × O3	59.1	(0.8)	64.5	(0.7)	93.3	(0.3)	216.0	(2.0)	70.4	(1.5)
29 Mar.–14 Nov.,	CF	4.9	(0.2)	5.2	(0.3)	8.8	(0.6)	25.5	(0.5)	0.0	(0.0)
2013 (231 days)	1.0 × O3	26.9	(0.3)	31.2	(0.4)	43.2	(0.9)	90.3	(3.6)	7.5	(0.6)
	1.5 × O3	47.1	(0.3)	49.5	(0.5)	69.4	(0.9)	143.3	(0.6)	34.9	(1.3)
	2.0 × O3	62.2	(0.3)	65.4	(0.2)	92.5	(1.1)	190.3	(2.5)	69.6	(0.9)

Each value is the mean of three chamber replicates, and the standard deviation is shown in parentheses. CF: charcoal-filtered air; 1.0 × O₃: 1.0 times the ambient O₃ concentration; 1.5 × O₃: 1.5 times the ambient O₃ concentration; 2.0 × O₃: 2.0 times the ambient O₃ concentration. ^a 12 h: 6:00–18:00. ^b Daylight AOT40: Accumulated exposure over a threshold of 40 nl l⁻¹ during daylight hours (global radiation > 50 W m⁻²).

shows the average O₃ concentrations and AOT40 of O₃ in each gas treatment during the O₃-exposure periods of 2012 and 2013. The exclusion efficiency of O₃ in the CF treatment was about 80%. Averaged across the two growing seasons, even though the seasonal mean O₃ concentrations in the 1.5× and 2.0×O₃ treatments were 1.62–1.74 and 2.19–2.33 times higher than that in the 1.0×O₃ treatment, respectively, the highest 1-h maximum O₃ concentration in the two treatments almost reached the target concentration (i.e., 1.53 and 2.02 times, respectively). The average concentration in 2012 was lower than that in 2013, whereas the daily 1-h maximum concentration was higher in 2012 than in 2013. Although this result suggests a stronger diel pattern in 2012, it was not obvious. The daylight AOT40 in the 1.0 × O₃ treatments in 2012 and 2013 were 6.0 and 7.5 ppm h and exceeded the concentration-based critical level for O₃-sensitive forest trees (5 ppm h) that was adopted by the United Nations Economic Commission for Europe Convention on Long-Range Trans-boundary Air Pollution [18].

To test the performance of stomatal conductance model for the leaves of F. crenata, Q. serrata, Q. mongolica var. crispula and B. platyphylla var. japonica seedlings, linear regression analyses were conducted between measured g_sw and g_sw estimated from the model of Kinose et al. (2014) [27], with modifications of g_max and the parameters of each function (

Table 3. Results of the linear regression analyses of the measured stomatal diffusive conductance to water vapor (g_sw, mmol H₂O m⁻² LA s⁻¹) and g_sw estimated from the model of Kinose et al. (2014), using the modifications of g_max and the parameters of each function based on the g_sw obtained in the present study.

	F. crenata	Q. serrata			Q. mongolica var. crispula			B. platyphylla var. japonica
	1st Flush (n = 351)	1st Flush (n = 312)	2nd Flush (n = 191)		1st Flush (n = 374)	2nd Flush (n = 166)		Early (n = 352)	Late (n = 662)
Slope	0.854	0.871	0.752		0.843	0.659		0.691	0.697
Intercept	29.0	40.5	88.4		72.4	135.2		113.7	253.1
R2 value	0.482	0.416	0.347		0.346	0.209		0.306	0.301
RMSE a	66.9	69.9	113.7		113.5	170.8		116.8	296.2
MBE b	7.1	29.8	61.5		30.3	60.5		71.8	185.1

^a RMSE: Root mean square error of the stomatal conductance model (mmol H₂O m⁻² LA s⁻¹); ^b MBE: Mean bias error of the stomatal conductance model (mmol H₂O m⁻² LA s⁻¹).

). The model parameterized in the present study accounted for 48.2% of the variation in the 1st flush leaves of F. crenata, 41.6% and 34.7% of the variation in the 1st and 2nd flush leaves of Q. serrata, respectively, 34.6% and 20.9% of the variation in the 1st and 2nd flush leaves of Q. mongolica var. crispula, respectively and 30.6% and 30.1% of the variation in the early and late leaves of B. platyphylla var. japonica, respectively. We did not evaluate the performance of stomatal conductance model using the g_sw and corresponding meteorological parameters obtained from the sites different from the present experiment site.

The relationships between WDM increments and the whole-plant-based ΣP_{n_est} during the two growing seasons are shown in Figure 1. The values are plotted for each species across all the treatments. There were significant positive correlations for F. crenata and Q. mongolica var. crispula but those for Q. serrata and B. platyphylla var. japonica were not significant.

The responses of relative A_sat or relative ΣP_{n_est} to POD₀ in the leaves of F. crenata, Q. serrata, Q. mongolica var. crispula and B. platyphylla var. japonica are shown in Figure 2a–h. The coefficients of determination (R²) for the response functions of relative A_sat or relative value of leaf-level ΣP_{n_est} to POD₀ were 0.663 and 0.894 in F. crenata, respectively, 0.645 and 0.935 in Q. serrata, respectively, 0.591 and 0.833 in Q. mongolica var. crispula, respectively and 0.602 and 0.760 in B. platyphylla var. japonica, respectively.

To discuss the species-specific responses of ΣP_{n_est} to POD, we examined the flux threshold appropriate for the linear regression of the responses of ΣP_{n_est} to POD_Y (

Table 4. The R² value for the linear regression line between relative value of leaf-level ΣP_{n_est} and phytotoxic O₃ dose (POD_Y, Y: flux threshold) in the leaves of F. crenata, Q. serrata, Q. mongolica var. crispula and B. platyphylla var. japonica seedlings.

Tree Species	No. of Plot	Y
		0	1	2	3	4	5	6	7	8	9	10
F. crenata	n = 8	0.710	0.747	0.805	0.850	0.881	0.903	0.917	0.921	0.918	0.903	0.878
		**	**	**	**	***	***	***	***	***	***	***
Q. serrata	n = 16	0.724	0.776	0.839	0.879	0.893	0.884	0.858	0.817	0.764	0.704	0.653
		***	***	***	***	***	***	***	***	***	***	***
Q. mongolica	n = 16	0.697	0.730	0.780	0.824	0.861	0.889	0.909	0.921	0.925	0.918	0.900
var. crispula		***	***	***	***	***	***	***	***	***	***	***
B. platyphylla	n = 36	0.738	0.754	0.768	0.775	0.774	0.767	0.755	0.740	0.722	0.703	0.681
var. japonica		***	***	***	***	***	***	***	***	***	***	***

Statistical significance for R₂ is shown: ** p < 0.01 and *** p < 0.001.

). The POD_Y appropriate for the linear regression of the response were POD₇ for F. crenata, POD₄ for Q. serrata, POD₈ for Q. mongolica var. crispula and POD₃ for B. platyphylla var. japonica.

In Figure 3, the relationships between the relative value of ΣP_{n_est} and that of ΣA_sat × days in the leaves of F. crenata, Q. serrata, Q. mongolica var. crispula and B. platyphylla var. japonica are shown. There was a significant positive linear correlation between the relative value of ΣP_{n_est} and that of ΣA_sat × days.

4. Discussion

In the present study, we evaluated the negative effects of O₃ on leaf-level photosynthetic CO₂ uptake, which could be easily incorporated into models of the carbon cycling, in the seedlings of four representative Japanese deciduous broad-leaved forest tree species based on POD. Lombardozzi et al. (2013) [39] compiled and analyzed the response of A_sat to POD using data from the peer-reviewed literature. Although the overall decrease in A_sat was observed, high variance masked any correlations between the decline in A_sat with increase in POD. Because POD could be related to cumulative O₃ damages, it could correspond not to O₃ effect on net photosynthetic rate at a certain time but to that on cumulative photosynthetic CO₂ uptake. Several researchers observed that the extent of the O₃-induced reduction in net photosynthetic rate varied seasonally [29,40]. On the other hand, Coleman et al. (1995) [41] observed a greater extent of O₃-induced reduction in net photosynthetic rate under high light intensities compared with under low light intensities, indicating variations in the extent of the O₃ damage on photosynthetic CO₂ uptake during the daytime and among canopy positions owing to diurnal variation in the light intensity and self-shading, respectively. In the present study, to take such seasonal and diurnal variations into account, we estimated ΣP_{n_est} from A-light curves measured monthly and diurnal changes in PPFD throughout the entire growing season. Because this estimation assumes that the A-light curves have been constant for about one month and ignored the diurnal changes in air temperature and relative air humidity, it was necessary to validate the estimation. For this purpose, we expanded the ΣP_{n_est} from a LA basis to the whole-plant basis by considering a roughly estimated LA per plant and whole-plant-level ΣP_{n_est} was compared with WDM increment over two growing seasons. Although not all the species showed significant correlations between WDM increment and the whole-plant-level ΣP_{n_est} during the two growing seasons (Figure 1), all the species had relatively high R² values. In general, whole-plant growth is largely determined by the amount of leaf area per plant and net photosynthetic rate per leaf area. Although the LA per plant was roughly estimated and calculation of ΣP_{n_est} did not consider the effects of environmental condition other than light intensity on photosynthetic CO₂ uptake, these results suggest that the leaf-level ΣP_{n_est} could reflect the cumulative photosynthetic CO₂ uptake of the seedlings per unit LA, which could be easily incorporated into models of the carbon cycling.

Because the negative impact of O₃ on photosynthetic CO₂ uptake can indirectly increase radiative forcing [7], we evaluated the effect of O₃ on photosynthetic CO₂ uptake based on POD₀ in the leaves of representative Japanese deciduous broad-leaved tree species. According to the meta-analysis by Wittig et al. (2007) [28], the 28% of variation in the O₃-induced percent changes in A_sat measured at a certain time was accounted for by the linear function of POD₀ (i.e., R² value was 0.28). On the other hand, Lombardozzi et al. (2013) [39] did not observe any correlations between changes in A_sat and POD owing to the large variability across studies. In the present study, about 60% of O₃-induced percent changes in the A_sat was accounted for by POD₀ (Figure 2a–d), which is greater than Wittig et al. (2007) [28]. However, the POD₀ accounted for the O₃-induced percent changes in the ΣP_{n_est} rather than those in the A_sat (Figure 2e–h). These results suggest that the ΣP_{n_est} is an appropriate dependent variable in the response function of photosynthetic CO₂ uptake to POD, which could result from the fact that the cumulative stomatal O₃ uptake corresponds to the extent of cumulative O₃ effect on photosynthetic CO₂ uptake. Therefore, the response function of relative leaf-level ΣP_{n_est} to POD is useful for the quantification of the negative impact of O₃ on the NPP of forests.

In the present analysis, there are uncertainties in the calculations of leaf-level ΣP_{n_est}, whole-plant-level ΣP_{n_est} and POD. For the calculation of leaf-level ΣP_{n_est}, the calculation period was from the first day of the first measurement of A-light curve to the last day of the last measurement in each growing season. Because this period was shorter than actual accumulation period, the leaf-level ΣP_{n_est} could underestimate the actual cumulative photosynthetic CO₂ uptake per unit LA. It is unclear that the ignorance of the effects of changes in air temperature and air humidity on the A-light curves causes overestimation or underestimation, but it would be the source of the error. Furthermore, it is also unclear the measurement of A-light curve at about one-month interval was enough time resolution to estimate the leaf-level cumulative photosynthetic CO₂ uptake. The greater the number of measurements during the growing season, the better for the estimation of absolute value of accumulated photosynthetic CO₂ uptake. In the present study, however, we focused on the extent of O₃-induced reduction in leaf-level ΣP_{n_est}, rather than absolute value. Because there were relatively high R² value in the relationship between WDM increment and the whole-plant-level ΣP_{n_est} in all tree species, the monthly measurement of A-light curve could be enough time resolution for the evaluation of O₃ effects on cumulative photosynthetic CO₂ uptake. For the scaling up ΣP_{n_est} from leaf-level to whole-plant-level, it is necessary to account for the difference in the light condition within the canopy and the changes in the leaf area per plant not only owing to the leaf emergence but also owing to the O₃-induced leaf abscission. The calculation of whole-plant-level ΣP_{n_est} did not consider the shelf-shading within the canopy and assumed that all leaves received full sunlight, which could result in the overestimation of the cumulative photosynthetic CO₂ uptake at whole-plant level. The estimation of changes in the leaf area per plant of tree species having flush-type leaf emergence, such as F. crenata, Q. serrata and Q. mongolica var. crispula, could be relatively easy. However, changes in the leaf area of B. platyphylla var. japonica having heterophyllous leaf emergence type is not easy to estimate, because it is difficult to identify the emergence day of each leaf, which could result in the higher variability than the other three tree species. The source of error in the calculation of POD could be estimation of g_sw in addition to the difficulty in the definition of leaf emergence day (i.e., start day of accumulation period) especially in B. platyphylla var. japonica. In the present study, the accuracy of estimation of g_sw was relatively low, especially for 2nd flush leaves of Q. mongolica var. crispula and early and late leaves of B. platyphylla var. japonica. On the other hand, mean bias error (MBE) in the relationship between estimated and measured g_sw were higher than 0 in all tree species (Table 3), indicating the overestimation of g_sw and thus resulting in overestimation of POD. Although we did not try adjusting other parameters to get a better fit, higher accuracy in the estimation of g_sw is foundational to calculating the POD. Further study is required to improve the accuracy of the estimation of g_sw by modification of the stomatal conductance model, considering the other parameters such as O₃-induced stomatal sluggishness (e.g., Hoshika et al., 2014 [42]).

Sitch et al. (2007) [7] quantitatively evaluated the O₃-induced reduction in global gross primary production (GPP) using the sensitivity to O₃ of the whole-plant growth of European beech, birch and oak and Norway spruce for forest trees. In their report, there was considerable difference in the O₃-induced reduction in GPP between the estimations considering a low or high plant O₃ sensitivity. Thus, an uncertainty in the estimation of the O₃-induced reduction in GPP could result from different sensitivities to O₃ among tree species. There are many forest tree species with species-specific O₃ sensitivity in Asia [32,33,43]. In the present study, the species-specific responses (i.e., O₃ sensitivities) of cumulative photosynthetic CO₂ uptake to stomatal O₃ uptake were clearly demonstrated (Figure 2e–h). In addition, the flux threshold appropriate for the linear regression of the responses of relative ΣP_{n_est} to POD_Y was also species-specific (Table 4). Therefore, the differences in the O₃-sensitivity among the representative temperate forest tree species should be taken into account for more accurate and quantitative evaluations of O₃ effects on photosynthetic CO₂ uptake.

According to a meta-analysis by Wittig et al. (2007) [28], the O₃ sensitivity of A_sat in the leaves of trees as assessed by the POD was 0.21% per mmol O₃ m⁻² LA. To compare the POD-based O₃ sensitivities, we conducted a linear regression analysis of the relationship between POD₀ and relative A_sat. The slopes of the regression lines for each tree species were −0.69, −0.95, −0.66 and −0.98% per mmol O₃ m⁻² LA for F. crenata, Q. serrata, Q. mongolica var. crispula and B. platyphylla var. japonica, respectively. These results indicate that the POD-based O₃ sensitivity of the A_sat of Japanese deciduous broad-leaved forest tree species is high compared with those of the tree species reported by Wittig et al. (2007) [28], although the POD-based O₃ sensitivity reported by Wittig et al. (2007) [28] varied greatly owing to differences in species, genotypes, locations and experimental conditions. Because it has been suggested that the degree of O₃ damage is determined by the balance between stomatal O₃ flux and leaf cellular detoxification [14,44,45], the higher POD-based O₃ sensitivity of the net photosynthesis in the leaves of Japanese deciduous broad-leaved tree species could be attributed to the lower detoxification capacity of O₃ in the leaves.

Among the four tree species used in the present study, there were obvious differences in the POD-based O₃ sensitivities of cumulative photosynthetic CO₂ uptake (Figure 2e–h). At a lower POD₀ up to around 20–30 mmol O₃ m⁻², ΣP_{n_est} of F. crenata, Q. serrata and Q. mongolica var. crispula were less responsive to increasing POD₀ as compared with that of B. platyphylla var. japonica which showed an almost constant reduction as POD₀ increased (Figure 2e–h). The extent of the pronounced plateau at a lower POD₀ suggests a tolerance to the O₃ taken into the leaves and is represented by the variable λ in the Weibull function [37], which were 3.07, 3.44, 2.80 and 1.78 in F. crenata, Q. serrata, Q. mongolica var. crispula and B. platyphylla var. japonica, respectively (Figure 2e–h). Matyssek et al. (2007) [46] supposed that leaf mass per area (LMA) reflects the depth of the biochemical defenses against O₃ taken into the leaves, and thus it could be used as an indicator of the leaf cellular detoxification capacity of O₃. In the present study, we measured LMA at the end of each growing season (data not shown). Although there was considerable variation in the LMA between the years and among the treatments within one species, there was a significant positive correlation between the λ and median LMA of each species (R = 0.970, n = 4, p < 0.05). This result suggests that, among the four tree species, different responses at a lower POD₀ might result from differences in detoxification capacity in the leaves.

Among the three tree species showing λ of around 3, the response of relative ΣP_{n_est} to increasing POD₀ of Q. serrata become drastically sensitive above the POD₀ of around 30 mmol O₃ m⁻². Because the activity of antioxidative enzymes and antioxidant concentration increases in response to O₃ exposure [15,47,48], the different responses of ΣP_{n_est} to increasing POD₀ above the POD₀ of around 30 mmol O₃ m⁻² might be caused by the difference in the inducible antioxidative capacity. On the other hand, differences in the flux threshold of POD_Y appropriate for the linear regression could also suggest differences in the detoxification capacity in the leaves among the tree species, because the flux threshold could focus the POD to the portion of the O₃ flux that is thought to be not detoxified in the leaves. However, the rank in descending order of the flux threshold was Q. mongolica var. crispula, F. crenata, Q. serrata and B. platyphylla var. japonica (Table 4) and did not match with that of the λ (Figure 2). The flux threshold of Y is the cut-off O₃ flux at a constant value throughout the growing season, even though the detoxification capacity in the leaves changes not only owing to O₃ exposure but also owing to leaf aging. Thus, the flux threshold of Y might not be biologically relevant. To make quantification of O₃ damage on photosynthetic CO₂ uptake more mechanistic and biologically relevant, therefore, further study is required to clarifying the mechanisms underlying the different response of ΣP_{n_est} to POD₀ among the species with emphasis not only on constitutive but also on inducible and phenological changes in antioxidative capacity in the leaves [14,15,44].

Although Lombardozzi et al. (2013) [39] did not observe any correlations between changes in A_sat and POD based on the data from the peer-reviewed literature, part of the literature could be useful for establishment of photosynthetic responses to POD, which could be easily incorporated into models of the carbon cycling. In the present study, O₃-induced reductions in net photosynthetic rate were greater under relatively high light intensities and tended to be similar above a PPFD of approximately 500 μmol m⁻² s⁻¹ (e.g., Figure S1), which was also observed in a study by Coleman et al. (1995) [41]. This result suggests that the O₃-induced reduction in A_sat considerably accounts for the reduction in the ΣP_{n_est}. Across the species and leaf types, we found a significant linear correlation, with a slope of 0.93, between the relative value of ΣP_{n_est} and that of ΣA_sat × days, which is parameter similar to seasonal mean percent reduction in A_sat as reported by Novak et al. (2005) [29] (Figure 3). Several data points, which were obtained from the leaves with a short cumulative period, such as the 2nd flush leaves of Q. serrata and Q. mongolica var. crispula and the late leaves of B. platyphylla var. japonica, were plotted slightly away from the 1:1 line. This could result from the low number of measurements taken during the accumulation period. Therefore, the A_sat, if measured at appropriate intervals throughout the growing season, could be used to evaluate the O₃-induced percent change in ΣP_{n_est}. Several researchers have reported such seasonal measurements of A_sat in the leaves of deciduous broad-leaved tree species [40,49,50,51]. These data sets could be used to estimate the degree of O₃-induced reduction in cumulative photosynthetic CO₂ uptake and its response to stomatal O₃ uptake, which is useful in the quantification of the negative impact of O₃ on the NPP of deciduous broad-leaved forest trees.

5. Conclusions

In the present study, the cumulative photosynthetic CO₂ uptake of four representative Japanese deciduous broad-leaved forest tree species was estimated from A-light curves measured periodically during the growing season. The whole-plant-level ΣP_{n_est} were highly correlated with the whole-plant dry mass increments over the two growing seasons. Because whole-plant growth is largely determined by the amount of leaf area per plant and net photosynthetic rate per leaf area, this result suggests that leaf-level ΣP_{n_est}, which was estimated from the monthly A-light curves and hourly PPFD, could reflect the cumulative photosynthetic CO₂ uptake of the seedlings per unit LA. The ΣA_sat×days calculated from periodically measured A_sat could be used to evaluate the O₃-induced percent change in leaf-level ΣP_{n_est}. Although the O₃-induced percent changes in leaf-level ΣP_{n_est} were well explained by POD₀ in all the tree species, species-specific responses of leaf-level ΣP_{n_est} to POD₀ were observed. In addition, the flux threshold appropriate for the linear regression of the responses of relative leaf-level ΣP_{n_est} to POD was also species-specific. Therefore, species-specific responses of cumulative photosynthetic CO₂ uptake to stomatal O₃ uptake could be used for more accurate evaluations of O₃ impact on the NPP of Japanese deciduous broad-leaved forest tree species.